中国林蛙卵核糖核酸酶的分离纯化及其抗肿瘤作用

A Cytotoxic Ribonuclease Isolated from Occytes of Chinese Northeast Frog(Rana dybowskii)

以中国林蛙卵为原料,采用丙酮分级沉淀、SP-Trisacryl阳离子交换色谱、SephadexG-75凝胶过滤色谱、C8反相色谱等纯化方法,得到一种具有核糖核酸酶活性的蛋白质,采用SDS-PAGE电泳对该蛋白质进行了相对分子质量和纯度测定。结果表明:纯化的中国林蛙卵核糖核酸酶为相对分子质量13kDa的单一成分。该酶最适反应温度为65℃,最适反应pH为5.5～6.0,米氏常数为4.11μmol/L,最大反应速率为2.82pmol/s。在体外细胞毒性实验中,对人三种肿瘤细胞HeLa、K562、MCF-7具有抑制作用,其IC50分别为0.6μmol/L、0.8μmol/L和4μmol/L,而对于正常人成纤维细胞在酶浓度达到8μmol/L时仍未见明显细胞毒性。这种从中国林蛙卵中分离纯化出的具有选择性细胞毒性的小分子量核糖核酸酶,为肿瘤的治疗提供了新的候选蛋白分子。

By acetone precipitation,SP-Trisacryl cation-exchange chromatography,Sephadex G-75 gel filtration chromatography and C8 reversed-phase chromatography,a single component ribonuclease is purified from oocytes of Chinese Northeast frog（Rana dybowskii）.SDS-PAGE electrophoresis shows a single band with relative molecular weight of 13kDa.Its optimum temperature is 65℃,and optimum pH is 5.5~6.0.Its Michaelis constant is 4.11μmol/L,and maximum reaction rate is 2.82pmol/s.In vitro cytotoxicity experiments the IC50 value for HeLa,K562 and MCF-7 tumor cells is 0.6μmol/L,0.8μmol/L and 4μmol/L separatedly,while for the normal human fibroblasts cells,even up to 8μmol/L,no cytotoxic effects were observed.This low molecular weight ribonuclease with selective cytotoxicity for tumor cells isolated from oocytes of the Chinese northeast frog（Rana Dybowskii）will join the candidate proteins of drug molecular for the treatment of cancer.